This application is filed under 35 U.S.C. xc2xa7371, and claims the benefit of the filing date under 35 U.S.C. xc2xa7119(a) for European Patent application serial no. EP 97121848.2, filed on Dec. 11, 1997, and claims the benefit of the filing date under 35 U.S.C. xc2xa7365 for PCT international application number PCT/EP98/07972, filed on Dec. 8, 1998.
The invention relates to a process for obtaining L-dihydroorotic acid (in the following xe2x80x9cL-DHOxe2x80x9d) by chromatography on an anionic exchange material in a base water mixture under a pressure from about 1.1 MPa to about 40 MPa. The process can be used to investigate the in vitro and in vivo activity of N-(4-trifluoromethylphenyl)-5-methylisoxazole-4-carboxamide, N-(4-trifluoromethylphenyl)-2-cyano-3-hydroxycrontonamide and similar compounds.
L-DHO can be determined by a silica gel chromatographic procedure with subsequent chemical derivatisation and colorimetric determination (Kesner, L., Aronson, F. L., Silverman, M., Chan, P. C., Clin. Chem 21/3 (1975) 353). Another method converts L-DHO enzymatically to orotic acid by L-dihydroorotic acid dehydrogenase (in the following xe2x80x9cDHODHxe2x80x9d) prepared from rat liver and, after chemical derivatization, detects orotate by colorimetric changes (Rogers, L. E., Nicolaisen, K., Experientia 28/10 (1972) 1259). The disadvantages of these methods are the interference of other materials in complex physiological solutions. In addition the mentioned procedures are very time consuming because of laborious sample preparation and therefore not applicable for routine analysis in large clinical studies.
In the endeavor to provide improved separation and isolation processes for obtaining L-dihydroorotic acid, it has now been found that the same can be achieved by chromatography of L-DHO in a base water mixture on an anionic exchange material and a pressure of about 1.1 MPa to about 40 MPa. The process can be used for the quantitative determination of L-DHO in cell lysates, mammalian serum and human serum. Said process is highly reproducible, sensitive and validated.
The invention, as it is explained in the claims, achieves the object by a chromatography process comprising the steps of:
a) obtaining a column comprising pressure-stable anionic exchange material;
b) loading the column with a sample solution including L-dihydroorotic acid;
c) performing chromatography;
d) eluting the L-dihydroorotic acid with an eluting solution containing a base water mixture;
said process being performed under a pressure from about 1.1 MPa to about 40 MPa.
The term pressure-stable anionic exchange material means, for example, materials such as macroporous (2,000 ) divinylbenzene/ethylvinylbenzene polymer or a microporous polyvinylbenzylammonium polymer cross-linked with divinylbenzene or mixtures thereof which are modified with alkanol quaternary ammonium; or vinylbenzylchloride/divinylbenzene macroporous polymer; or crosslinked Polyethyliminopolymer; or silica modified with propyl-trimethyl-ammonium; or poly(styrene-divinylbenzene)trimethylammonium. The following products are particularly preferred:
Ion Pac AS 11, CarboPac PA 1 or CarboPac MA 1 anion exchange columns supplied by Dionex Corporation, Idstein, Germany,
GROM-SIL, Strong Anion. or GROM-SIL, Weak Anion.; supplied by Grom
P1000 SAX, Ionospher SA or Chrompack PA; supplied by Chrompack
PRP-X100 or RCX-10 supplied by Hamilton
The election solution contains a base water mixture. Suitable bases are derived from alkali metals or alkaline earth metals, such as sodium hydroxide, potassium hydroxide, magnesium hydroxide or calcium hydroxide. The concentration of the base is from 1 mmol/l to about 200 mmol/l, based on water as solvent, preferably from 2 mmol/l to about 120 mmol/l, particularly preferred is 100 mmol/l. The temperature during the chromatography procedure is from about 0xc2x0 C. to about 50xc2x0 C., preferably from about 15xc2x0 C. to about 30xc2x0 C., particularly preferred from about 19xc2x0 C. to about 25xc2x0 C. The operating pressure during the chromatography is substantially constant. The chromatography can be carried out using different pressures, for example the chromatography can be carried out under a pressure from about 1.1 106 Pa (1.1 MPa) to about 40 106 Pa (40 MPa), in particular from 4.1 MPa to 5.5 MPa. The eluent flow rates are from about 0.2 ml/min to about 3 ml/min, preferably 1 ml/min. The loading of the columns, chromatography, and elution of the L-DHO takes place by known, conventional technical methods. A suitable elution is one in which the elution displays a time gradient of the base concentration, preferably with a linear course. This concentration gradient can be applied, for example, by a low base concentration (zero in the limiting case) being present in the elution at the start of the elution, and by increasing the base concentration during the elution process. It is possible in this way to achieve a particularly effective separation of the L-DHO in samples derived from serum or cell lysates. A preferred base gradient varies from near 1% NaOH (100 mmol/l) and 99% water (at the start of the elution) to about 60% NaOH and 40% water (at the end of the elution), with the particular preferable range being from about 1% NaOH and 99% water (at the start of the elution) to about 15% NaOH and 75% water (at the end of the elution). The base water gradient is changed in a linear way from 2.5 min to about 14 min and from 14 min to about 25 min, wherein the steep of the gradient is different during these 2 time periods.
A particularly suitable elution can be achieved by using a low base concentration at the start of the separation process of about 1% for a time period of about 2.5 minutes. The result is eluting most of the interfering material from the biological matrix from the column. The separation of the analyte is achieved by slowly increasing the gradient to about 23% of base within a time period of a 14 minutes total analysis time. Then the base cncentration is incresed to about 60% within 4 min to allow the elution of strongly bond material. 60% base should be applied for no longer than 6 min until reequilibration is performed by 1% base water mixture. The next analysis is started after 45 min of total analysis time. The water in the base water gradient has to be deionized and degassed.
The separation process according to the invention takes place in a column process. The temperature, which is preferably kept constant during the anionic exchange chromatography, may be varied within a wide range. A preferred temperature range is from about xe2x88x9210xc2x0 C. to about 50xc2x0 C., in particular from about 15xc2x0 C. to about 25xc2x0 C.
The elution of L-DHO takes place from 10 min to 12 min after start of the gradient. The running time of the elution process is from 13 min to 25 min. The L-DHO is detected by a conductivity detector, such as model CD20 from Dionex Cooperation. In order to minimize the base line shift and to lower the background conductivity an anion self regeneration suppresser, such as model ASRS-I, 4 mm from Dionex Cooperation, can be used.
The process according to the invention is in particular suitable for analytical chromatography but can also be used for preparative chromatography, especially when the process according to the invention is carried out with a preparative high pressure liquid chromatography column (HPLC) system. The term xe2x80x9cpreparative chromatographyxe2x80x9d means a purification process with the aim of obtaining, and not merely analyzing, pure products. The amount of the pure products can vary within wide limits, for example from 1 mg to 1000 g, preferably between 50 mg and 500 mg.
The process according to the invention can be used to detect changes in intracellular or extracellular L-DHO concentrations due to the inhibition of dihydroorotic acid dehydrogenase (DHO-DH). The enzyme DHO-DH is responsible for the conversion of L-dihydroorotic acid to orotic acid during the de novo pyrimidine synthesis. The inhibition of DHO-DH leads to the accumulation of L-DHO. The process according to the invention can be used for the preparation of a diagnostic assay. The process according to the invention can be used for determining the activity of DHO-DH inhibitors. DHO-DH inhibitors are for example N-4-(trifluoromethylphenyl)-5-methylisoxazol4-carboxylamide, Brequinar, N-(4-(trifluoromethylphenyl)-2-cyano-3-hydroxyhept-2-en-6-yne-carboxamide, 2-cyano-3-cyclopropyl-3-hydroxyacrylic-acid-(4-cyanophenyl)-amide or N-(4-trifluoromethylphenyl)-2-cyano-3-hydroxycrotonamide. The process according to the invention can be used for determining L-DHO concentrations in plants, cell lines, animals and human beings. L-DHO determination can be used to monitor the activity of DHO-DH inhibitors in plants, mammals and human beings.
The process according to the invention is described in detail in the examples which follow. Unless otherwise indicated, percentage data relate to weight.